G C A T T A C G G C A T genes Article The Chloroplast Genome of Symplocarpus renifolius: A Comparison of Chloroplast Genome Structure in Araceae Kyoung Su Choi ID , Kyu Tae Park and SeonJoo Park * ID Department of Life Sciences, Yeungnam University, Gyeongsan 712-749, Gyeongsangbuk-do, Korea; [email protected] (K.S.C.); [email protected] (K.T.P.) * Correspondence: [email protected]; Tel.: +82-53-810-2377 Academic Editor: Charles Bell Received: 5 September 2017; Accepted: 6 November 2017; Published: 16 November 2017 Abstract: Symplocarpus renifolius is a member of Araceae family that is extraordinarily diverse in appearance. Previous studies on chloroplast genomes in Araceae were focused on duckweeds (Lemnoideae) and root crops (Colocasia, commonly known as taro). Here, we determined the chloroplast genome of Symplocarpus renifolius and compared the factors, such as genes and inverted repeat (IR) junctions and performed phylogenetic analysis using other Araceae species. The chloroplast genome of S. renifolius is 158,521 bp and includes 113 genes. A comparison among the Araceae chloroplast genomes showed that infA in Lemna, Spirodela, Wolffiella, Wolffia, Dieffenbachia and Colocasia has been lost or has become a pseudogene and has only been retained in Symplocarpus. In the Araceae chloroplast DNA (cpDNA), psbZ is retained. However, psbZ duplication occurred in Wolffia species and tandem repeats were noted around the duplication regions. A comparison of the IR junction in Araceae species revealed the presence of ycf1 and rps15 in the small single copy region, whereas duckweed species contained ycf1 and rps15 in the IR region. The phylogenetic analyses of the chloroplast genomes revealed that Symplocarpus are a basal group and are sister to the other Araceae species. Consequently, infA deletion or pseudogene events in Araceae occurred after the divergence of Symplocarpus and aquatic plants (duckweeds) in Araceae and duplication events of rps15 and ycf1 occurred in the IR region. Keywords: Araceae; Symplocarpus; infA; psbZ; inverted repeat region; phylogenetic relationship 1. Introduction The family Araceae consists of approximately 102 genera and 3300 species and is one of most structurally diverse groups of monocots. This family is widely distributed in all the continents, except Antarctica. The Lemnoideae subfamily (duckweeds) within Araceae displays particularly diverse morphological traits, habits and life forms [1–3]. The earliest modern classification of Araceae was given in 1860 by Schott [4] and Engler [5,6]. They classified Araceae according to the vegetable morphology and anatomy. Many molecular phylogenetic analyses support this classification. However, the taxonomic relationships among some species have not been resolved, for example, among the members of Aroideae subfamily [2,3,7,8]. The genus Symplocarpus belongs to the subfamily Orontioideae, which also contains the genera Orontium and Lysichiton [9]. Five Symplocarpus species (S. renifolius Schott ex Miq., S. foetidus (L.) Nutt., S. nipponicus Makino, S. nabekuraensis Otsika and K.Inoue and S. egorovii N.S. Pavlova and V.A. Nechaev) are distributed in eastern Asia and eastern North America [9–11]. The plant chloroplast (cp) genomes are divided into four major segments. Two of these contain only single copies of genes: the large single-copy (LSC) region and small single-copy (SSC) region. The last two segments are nearly identical inverted copies, termed inverted repeats (IRA and IRB). Genes 2017, 8, 324; doi:10.3390/genes8110324 www.mdpi.com/journal/genes Genes 2017, 8, 324 2 of 15 The cp genomes are approximately 120–170 kb and include 100–120 genes. Most angiosperm cp genomes are highly conserved [12,13]. However, there have been observations of gene loss [14], inversion [15], rearrangement [16], IR loss [17], IR contraction and IR extension [18] in some lineages. In particular, four junctions (LSC/IRB, IRB/SSC, SSC/IRA and IRA/LSC) of cp genome were found at various sites. Wang et al. [18] suggested two possible mechanisms, (i) gene conversion to account for the small IR expansion or movements in most species of the genus; (ii) a DNA double-strand break (DSB) to explain the extensive incorporation of the LSC region. We observed that the IR region has expanded in three species of Lemnoideae. Previous studies on the cp genomes in the Araceae family have focused on five species. Mardanov et al. [19] studied the cp genome of Duckweed (Lemna minor) and compared it with those of the other angiosperm species. Wang and Messing [20] generated the cp genomes of three other duckweed species (Spirodela polyrhiza, Wolffiella lingulata and Wolffia australiana) and Ahmed et al. [21] determined the cp genome sequences for two taro species (subfamily Aroideae) and compared the Aroid cp genomes. In this study, we determined the complete cp genome sequence of the Asian skunk cabbage Symplocarpus renifolius (subfamily Orontioideae, family Araceae) and compared it with those of other Araceae species. We also analyzed the gene contents (infA and psbZ), IR region extensions (ycf1 and rps15 duplication) and phylogenetic relationships in Araceae. 2. Materials and Methods 2.1. Plants Materials and DNA Sequencing This study was approved by the Korean National Arboretum (KNA 1-2-13, 14-2). The S. renifolius sample was obtained from a living collection in the greenhouse of Yeungnam University, in Gyeongsan, South Korea. Total DNA was extracted using a DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA, USA) and was quantified using a HiGen Gel and PCR Purification system (Biofact Inc., Daejeon, Korea). The genomic DNA was sequenced using Illumina Miseq (Illumina Inc., San Diego, CA, USA). A total number of 4,491,905,669 raw reads were obtained from the Illumina sequencing. These raw reads were trimmed and filtered using Genious v.10.1 (Biomatters, Auckland, New Zealand). The filtered 1,516,987 reads were mapped to reference genome, Dieffenbachia sequine (NC_027272). The coverage of S. renifolia was 1002 X. The genome coverage was estimated using the CLC Genomics Workbench, v. 7.0.4 (CLC Bio, Aarchus, Denmark) at LabGenomics Co. (Seongnam, Korea). 2.2. Genome Annotation, Genome Mapping and Sequence Analysis The complete cp genome sequence was annotated using Dual Organellar GenoMe Annotator (DOGMA) [22] and was compared with the published complete cp genome sequences (Table S1) available for Colocasia esculenta (JN105689), Dieffenbachia seguine (NC_0272725), Spirodela polyrhiza (JN160603), Wolffia australiana (JN160605) and Wolffiella lingulata (JN160604). All the identified tRNA genes were verified using the corresponding structures predicted by tRNAscan-SE [23]. A circular cp genome map was drawn using ORDRAW [24]. To compare the structure and genes present in Araceae family, sequences from the different plants were aligned using MAFFT [25] and Geneious v.6.1.7 bioinformatics software platform (Biomatters). 2.3. Repeat Structure Tandem repeats (forward, palindrome, reverse and complement repeats) were detected using REPtuer program [26]. The minimum repeat size was set at 30 bp and at a sequence identity greater than 90%. The simple sequence repeats (SSRs) were detected using Phobos v.3.3.12 (http://www.ruhr- uni-bochum.de/ecoevo/cm/com_phobos.htm). We applied a threshold 10 to mononucleotide repeats, five to dinucleotide repeats, four to trinucleotide repeats, three to tetranucleotide repeats and two to penta-, hexa-, hepta- and deca- nucleotide repeats. Genes 2017, 8, 324 3 of 15 2.4. Phylogenetic Analysis A total of 77 coding genes from 17 species were compiled into a single file of 67,392 bp and aligned using MAFFT [25]. Sixteen species (including six Araceae species) were selected as the in-groups and Amborella trichopoda was included as the out-group (Table S1). Maximum likelihood (ML) analyses were performed using RAxmL v.7.4.2 with 1000 bootstrap replications and using the GTR+I+G model [27]. The best-fit models of substitutions selected by ModelTest 3.7 [28]. 3. Results 3.1. Characteristics of the Symplocarpus renifolius and Araceae cp Genomes The cp genome of S. renifolius (GenBank accession number KY039276) is 158,521 bp in length (Figure1). It includes two inverted repeat regions (IR A and IRB) of 25,801 bp separated by LSC and SSC regions of 86,620 bp and 20,299 bp, respectively. The overall AT content of the S. renifolius cp genome is 62.7% (Table1). The S. renifolius cp genome contained 113 genes, of which four were ribosomal RNA (rRNA) genes, 30 were transfer RNA (tRNA) genes, 78 genes were protein coding genes and 17 genes located in the IR regions were duplicated (rpl2, rpl23, trnI-CAU, ycf2, trnL-CAA, ndhB, rps7, rps12, trnV-GAC, rrn16, trnI-GAU, trnA-UGC, rrn23, rrn4.5, rrn5, trnR-ACG and trnN-UGA). Of the 113 single-copy genes, 13 contained a single intron (eight protein-coding genes and five tRNA genes) and three protein-coding genes (ycf3, clpP and rps12) contained two introns. The seven complete cp genomes of Araceae species (Lemna, Spirodela, Wolffiella, Wolffia, Dieffenbachia, Colocasia and Symplocarpus) consist of a pair of IRs (25,273–31,930 bp) separated by one LSC (86,670–92,015 bp) and one SSC (13,394–22,208 bp) region, each. S. renifolius has one of the smallest cp genomes and has the lowest AT content among the Araceae species (Table1). Table 1. General features of Symplocarpus and comparison to other Araceae species. Symplocarpus Colocasia Dieffenbachia Lemna Spirodela Wolffiella Wolffia Total length 158,521 bp 162,424 bp 163,699 bp 165,955 bp 168,788 bp 169,337 bp 168,704 bp LSC 86,620 bp 86,670 bp 90,780 bp 89,906 bp 91,222 bp 92,015 bp 91,454 bp SSC 20,299 bp 22,208 bp 22,440 bp 13,603 bp 14,056 bp 13,956 bp 13,394 bp IR 25,801 bp 25,273 bp 25,235 bp 31,223 bp 31,755 bp 31,683 bp 31,930 bp % AT content 62.7% 63.8% 63.6% 64.3% 64.3% 64.2% 64.1% Genes Coding genes 80 79 79 79 79 79 79 tRNA 30 30 30 30 30 30 30 rRNA 4 4 4 4 4 4 4 IR duplication 17 17 17 17 19 19 19 genes LSC: Large Single Copy, SSC: Small Single Copy, IR: Inverted Repeat, tRNA: transfer RNA, rRNA: ribosomal RNA.